梯度稀释及微量点样法在环境γ-变形菌分离中的应用

[目的]细菌分离中,梯度稀释及微量点样法可较好地保持细菌的原位丰度,理论上对优势菌的分离有较高的概率;应用梯度稀释及微量点样法分离γ-变形菌是一个有趣的研究点。[方法]应用梯度稀释及微量点样法,结合不同的分离条件,对7种环境样品进行γ-变形菌分离。[结果]从7种环境样品中成功分离52株细菌,经鉴定γ-变形菌为42株,占分离细菌总数的80.77%,呈现明显的γ-变形菌偏好性;已获得的γ-变形菌分属于8个不同的属,优势菌属为Klebsiella、Enterobacter、Escherichia,占已分离γ-变形菌的78.58%。[结论]实验所应用的梯度稀释及微量点样法对γ-变形菌的分离呈明显的偏好性(P0.05),是环境γ-变形菌分离方法的重要补充,该方法适用于环境优势菌的分离尝试。     

抗菌肽抑菌活性测定方法的研究

探讨建立一种在抗茵肽分离、纯化过程中简单、快速、灵敏的检测抑菌活性的方法。以微量稀释法为基础,利用氯化三苯四氮唑的显色原理设计了简单的显色MIC方法,且用该方法与K—B以及常规微量稀释法做了3种标准菌株的对比。与K—B法及常规微量稀释法相比,TTC微量稀释法可以准确地判断细菌生长抑制终点,得到确切的结果。TTC微量稀释法反应灵敏．操作简便,尤其适合在分离、纯化过程中跟踪检测抗菌肽的活性。     

土壤中辣椒疫霉分离方法的研究与量化测定

从杭州、西安、广州及武汉等辣椒病田分别采集土样 ,室内晾干研碎后 ,用选择性培养基 ,采用土壤稀释平板法和组织诱饵法分离辣椒疫霉 (PhytophthoracapsiciLeonian) ,并对土壤中辣椒疫霉的密度进行量化处理。结果表明 ,利用选择性燕麦培养基 ,采用土壤稀释平板法可分离获得大量的辣椒疫霉菌株 ,而且辣椒连作田的辣椒疫霉菌密度高于轮作田。组织诱饵法试验结果表明 ,辣椒叶片诱集效果最好 ,其次是辣椒果实。     

鸡卵黄抗体IgY的分离纯化及鉴定

采用水溶稀释法结合硫酸钠二次盐析沉淀法分离纯化鸡卵黄中蛋白IgY,实验中比较了不同pH值的水溶稀释液对卵黄除脂效果的影响;并采用SDS-PAGE及western blotting对提取产物进行鉴定。结果显示,pH值5.2水溶稀释液除脂效果最好,IgY得率最高。实验优化了鸡卵黄抗体IgY分离纯化技术,得到的IgY产量高、纯度高,特异性强;此外,水溶稀释法制备IgY具有利用小体积样品获得大量蛋白及纯化效率高的优点。     

盐度对稀释平板法研究红树林区土壤微生物数量的影响

在使用稀释平板法分离潮间带红树林及其对照光滩土壤微生物以及计数时,多数情况下使用陈海水制作培养基和稀释水,很少考虑培养基和稀释水的盐度对最终计数结果的影响.使用稀释平板法研究了盐度对福建九龙江口红树林区与深圳福田红树林保护区土壤微生物平板计数的影响,结果表明培养基与稀释水盐度对微生物数量有明显的影响.统计分析显示细菌的海水稀释效果优于淡水,而放线菌与真菌则刚好相反(P<0.05,一个例外).海水不适合配制红树林区土壤微生物平板计数的培养基,从0～35,高盐度的平板培养基会降低微生物的数量,尤其是放线菌的数量,尽管培养基的盐度对真菌影响无规律,但细菌数量在低盐度时比在高盐度和不加氯化钠时要多.根据盐度效应,提出了稀释平板技术应用于潮间带的红树林及其相应光滩时的优化方法,认为细菌应该用海水作无菌稀释水,而放线菌和真菌则应用淡水作稀释水;包括光滩在内的红树林区土壤微生物分离与计数的培养基宜控制较低盐度范围.     

嗜麦芽窄食单胞菌对四环素类抗生素药敏试验评价

目的了解琼脂扩散法（K-B法）及肉汤稀释法检测嗜麦芽窄食单胞菌的耐药性,了解两种方法的差异及为临床分离的嗜麦芽窄食单胞菌提供药敏结果。方法对28株临床分离嗜麦芽窄食单胞菌进行K-B法及肉汤稀释法检测,了解扩散直径及每株菌的MIC值。结果多西环素、米诺环素对嗜麦芽窄食单胞菌的药敏结果较好;K-B法及肉汤稀释法所得结果相关性好。结论临床上可以选择多西环素、米诺环素治疗嗜麦芽窄食单胞菌感染;可以应用K-B法检测嗜麦芽窄食单胞菌对四环素类抗生素的药物敏感性。     

八角炭疽病拮抗放线菌RX2-2的分离鉴定及其生物活性评价

[背景]八角炭疽病主要是由哈锐炭疽菌(Colletotrichum horii)引起的真菌性病害,给八角产业带来严重的经济损失.[目的]从八角根系土壤中分离筛选对哈锐炭疽菌具有拮抗作用的放线菌菌株,并对其进行种属鉴定及抗菌活性评价.[方法]采用稀释涂布平板法分离放线菌,并以哈锐炭疽菌作为指示菌,利用平板对峙法筛选具有高...     

ε-聚赖氨酸发酵过程污染微生物的分离与鉴定

【目的】研究ε-聚赖氨酸发酵过程中污染微生物的种类。【方法】采用稀释涂布法、划线法、环境胁迫法和液体营养富集法等对污染样本进行微生物的分离与纯化,通过菌落形态和显微观察,再结合16S rRNA基因序列分析,确定分离菌株的系统发育地位,并对分离菌株的ε-聚赖氨酸耐受性进行考察。【结果】液体营养富集法实现了污染微生物的分离,通过16S rRNA基因序列分析鉴定其为一株Acinetobacter bereziniae,并证实该菌能在高浓度ε-聚赖氨酸条件下生长。【结论】Acinetobacter bereziniae是ε-聚赖氨酸发酵过程中的主要污染微生物,这为后期发酵污染防治提供了一定的指导作用。     

广西北部湾放线菌的分离筛选及活性产物的鉴定

为从广西北部湾的泥样和植物中分离海洋放线菌,筛选具有抑菌活性的菌株,分离活性化合物。研究采用普通稀释法分离菌株,对发酵产物进行抑菌活性测试,利用活性追踪分离活性化合物,并通过波谱方法确定化合物结构。结果表明从6个海泥样品和3个植物样品中共分离73株放线菌,筛选得到具有抑香蕉枯萎病和金黄色葡萄球菌活性的菌株7株,并从其中的1株链霉菌 Streptomyces sp.MDCW-126的次级代谢产物中分离鉴定了星形孢菌素。从广西北部湾分离的药用活性菌株资源具有开发和深入研究价值。     

毛细管束法分离土壤稀有放线菌

描述了一种改进的从土壤中分离稀有放线菌的毛细管法。本法能在较低的稀释条件下,利用放线荫孢子和孢囊的疏水性、游动孢子在水中的运动性和趋化性(包括趋氧性)。从而大大提高稀有放线菌的检出率。     

洗发、护发化妆品微生物检查方法学研究

为提高化妆品潜在微生物的阳性检出率,建立洗发、护发类化妆品微生物限度和控制菌检查方法。采用常规法、培养基稀释法、薄膜过滤法对4种洗发、护发类化妆品进行微生物限度与控制菌方法学研究。结果显示,飘柔长效柔顺滋养洗发露、海飞丝去屑洗发露、飘柔人参滋养润发精华素、力士密集滋养修复-发膜级精华素菌落总数检测方法分别为0.2 m L/皿法、800 m L/膜法、0.5 m L/皿法和300 m L/膜法;霉菌及酵母菌检测方法分别为300 m L/膜法、300 m L/膜法、1 m L/皿法和1 m L/皿法;除海飞丝去屑洗发露采用培养基稀释法,其他均采用常规法进行控制菌检查。建议在化妆品微生物检验前应进行微生物方法研究,从而提高化妆品"潜在"病原菌的检出率。     

多菌种酸奶中活性乳酸菌的计数方法研究

利用4种选择性培养基MRS、MRS-山梨醇、MRS-5．2、Elliker,采用平板涂布法和倾注接种法。在蜡烛缸法（缺氧）、封口膜法（微氧）和供氧条件下对4种市售多菌种酸奶中保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌进行选择性计数方法研究。结果表明：蜡烛缸法较能反映乳酸菌活菌数量的实际情况,封口膜法次之;在不同培养条件下4种选择性培养基对于乳酸菌活菌的计数都是灵敏的;而涂布法和倾注法接种活菌数有显著差异,倾注法要优于涂布法。     

一种简单快速高分辨率的PAGE胶显带方法

尽管使用常规方法对聚丙烯酰胺胶(PAGE胶)进行DNA显带, 能获得高分辨率图片, 但常规方法操作步骤繁琐, 耗时较长。文章报道了一种PAGE胶DNA显带的改进方法, 分别使用改进的显带方法和常规的显带方法进行了PAGE胶的显影和比较。结果表明, 使用改进的显带方法得到的PAGE胶图片, DNA带型和背景具有更高的对比度, 因此具有更高的分辨率; 同时操作步骤少, 耗时短, 使用试剂少。这种方法在本实验室中已经完全代替了常规PAGE胶显带方法。     

酵母双杂交相关方法的改良及应用

对酵母双杂交实验过程中较为耗时的阳性克隆鉴定过程进行改进,以期建立一种快速有效的鉴定方法。分别采用液氮冻融法、超声破碎法、渗透压破壁法以及煮沸裂解法裂解酵母细胞,获得质粒作为PCR模板,直接测序鉴定筛选到的相互作用蛋白。以液氮冻融法和超声破碎法裂解细胞获得的质粒为模板进行PCR,得到特异的产物,测序鉴定结果明确,与经典的鉴定方法相比效果相当,但更加经济快捷;而渗透压破壁法和煮沸裂解法则效果不好。说明前两种方法可代替常规方法用于阳性克隆的鉴定,从而加快酵母双杂交实验中大量阳性克隆的筛查工作。     

Genotyping of single nucleotide polymorphism using model-based clustering

MOTIVATION: Single nucleotide polymorphisms have been investigated as biological markers and the representative high-throughput genotyping method is a combination of the Invader assay and a statistical clustering method. A typical statistical clustering method is the k-means method, but it often fails because of the lack of flexibility. An alternative fast and reliable method is therefore desirable. RESULTS: This paper proposes a model-based clustering method using a normal mixture model and a well-conceived penalized likelihood. The proposed method can judge unclear genotypings to be re-examined and also work well even when the number of clusters is unknown. Some results are illustrated and then satisfactory genotypings are shown. Even when the conventional maximum likelihood method and the typical k-means clustering method failed, the proposed method succeeded.     

Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods

To address the issue of excess polyethylene glycol (PEG)-lipid degradation observed when PEG-modified liposomes are prepared using the pH-gradient method, a concept using a novel PEG-modification method, called the post-modification method, was proposed and evaluated. To assess the proof concept, a preservation-stability study and a pharmacokinetic study were performed that compared the conventional PEG-modification method, called the pre-modification method, with the post-modification method. The results show that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, the post-modification method could be used without any manufacturing process difficulties, especially with high PEG-lipid content. In addition, a higher blood circulation capability was observed in the post-modification method. Through comparative studies, it was found that the post-modification method was advantageous compared to the pre-modification method. In conclusion, the post-modification method has the potential to be a novel PEG-modification method that can achieve a higher preservation stability of PEG-lipid, a greater ease of manufacturing, and a higher blood circulation capability, especially in the manufacturing of pH-gradient liposomal products.     

炭末明胶改良安瓿法应用研究

为了证实炭末明胶改良安瓿管法替代其他方法检测细菌是否产明胶酶,实验中采用营养明胶法、X线胶片法和炭末明胶改良安瓿管法对486株质控菌株、临床分离菌株进行明胶液化试验。结果显示,以营养明胶法培养的359株呈阳性反应,X线胶片法274株呈阳性反应,炭末明胶改良安瓿法423株呈阳性反应。营养明胶法明胶液化平均天数为3.3d,而炭末明胶改良安瓿法明胶液化平均天数仅为1.5d。炭末明胶改良安瓿法由于简单快捷,易于观察,而且试剂用量小、能够室温长期保存,该方法值得推广应用。     

A weighted test-area method for calculating surface tension

In this paper, we propose a weighted test-area method to calculate surface tension by incorporating the weighting factor from the Bennett method into the free energy perturbation scheme of the test-area method. This new method was tested by comparing against the results of the Bennett and test-area methods for simulations of square well (SW), Lennard-Jones and point charge fluid models. It is seen that the new method is accurate for all these simulations, giving the same results as the Bennett method, in contrast to the test-area method which cannot calculate the surface tension of a SW fluid. The new method converges as quickly, on the basis of computational time required, as the test-area method and almost twice as quickly as the Bennett method. This combination of speed and accuracy means that the weighted test-area method should be used in preference to the test-area method and Bennett method for surface tension and other macroscopic thermodynamic quantities that can be calculated through perturbation methods.     

演化极端结合分支分类方法

从生物演化的逆方向考虑,提出一种聚合的分支分类运算方法,称为演化极端结合分支分类法。文章阐明其设计思路、演算步骤,并以实例具体说明其演算过程。最后以演化长度系数、合理解与合理方法等概念,对演化极端结合法进行评价。     

The neighbor-joining method: a new method for reconstructing phylogenetic trees

A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.